The goal of this proposal is to explore ways to generate a novel AAV vector to overcome the barrier of pre- existing vector neutralizing antibodies (NAbs) to achieve efficient gene transfer in vivo. In vivo delivery of AAV vectors has shown promise in a variety of pre-clinical and clinical models of inherited disorders. The major challenges to date involve host immune responses and poor transduction efficiency due to limited vector tropism and poor delivery of the vector to the target cell. AAV serotype 8 (AAVS) has been identified as the best Clinical Candidate vector for liver-directed gene transfer and is currenfiy being evaluated in a human clinical trial for gene therapy of hemophilia B. Much is still being learned about host-vector immune interactions such as the effect of innate immunity and activation of T cells and B cells against the vector and its transgene product. One of the most important aspects of host immunity is the impact of neutralizing antibodies (NAb) on transduction efficiency. NAb against the AAV vector capsid results in substantial reduction in transducfion when administered systemically, as demonstrated in preclinical studies in animal models and in human clinical trials. We have characterized the threshold titer of pre-existing NAb that is compromising to AAVS gene transfer in nonhuman primates. Pre-existing NAb fiters in excess of 1:10 substantially diminish hepatocyte transduction at the dose of 3x10^^ genonne copies/kg. Based on our survey in human samples, we predict that 25% of humans in the USA will not be suitable for systemic delivery of AAVS vector. Engineered vectors that can escape neutralization would provide a more general and effective solufion. In this project, we will employ mulfiple approaches guided by AAV structures with the ultimate aim to generate an AAVS-prime (AAVS') vector that retains the high liver tropism of AAVS and is more resistant to neutralization in humans. RELEVANCE (See instructions): Many inherited metabolic diseases, such as OTC deficiency, are lacking effective treatments. Adeno- associated vector-mediated hepatic gene therapy has the potential to provide an alternative to cure or treat these life-threatening disorders. This project aims to developing improved vectors to overcome the barriers to achieving efficient therapeutic effects.